Compositions for treating bone defects

ABSTRACT

In certain described embodiments, implantable medical materials comprise a scaffolding material, a liquid organic binder, and entrapped calcium-containing particles. The medical materials can incorporate an osteoinductive factor such as a protein. The scaffolding material can bind the factor. In additional described embodiments, implantable medical materials include an osteoconductive scaffolding material, an incorporated osteoinductive factor, and a biodegradable barrier material effective to delay release of the factor from the scaffolding material. The scaffolding material can bind the factor. Also described a methods for preparing and implanting the described medical materials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/949,990, filed Dec. 4, 2007, which is incorporated herein byreference, in its entirety.

BACKGROUND

The present invention relates generally to medical implant compositions,and in certain aspects to malleable medical implant materials, such asputties, useful as carrier materials and/or bone graft materials.

A variety of materials have been suggested for the treatment of bonedefects. In addition to traditional bone grafting, a number of syntheticbone graft substitutes have been used or explored, including severalputty materials. To conduct bone through-growth effectively, implantmaterials derive benefit from the presence of substantial scaffoldingmaterial. Such scaffolding material must be combined in a compositionthat handles effectively during and after implant and in the case ofcarriers for growth factors such as morphogenetic proteins, the overallcomposition benefits from an effective utilization of the protein topromote or induce tissue growth through the scaffolding material.

In view of the background in the area, there exist needs for improvedputty materials which maintain the desired combination of malleabilityand cohesiveness as well as exhibit the ability to conduct bone growthbased upon native signals at the implant site and/or to effectivelyadminister an added signal molecule at the implant site. In certainaspects, the present invention is directed to these needs.

SUMMARY

In certain aspects, the present invention relates to malleable medicalimplant formulations that include a noncollagenous scaffolding materialcombined with a biocompatible liquid organic binder. Accordingly, incertain embodiments, the invention provides an osteoinductivecomposition comprising a malleable, mechanically entangled mass ofelongate, compliant noncollagenous scaffolding elements. The compositioncomprises sulfated glycosaminoglycan molecules bound to the scaffoldingelements and a liquid organic binder that coats the scaffoldingelements, wherein the liquid organic binder has a viscosity and ispresent in an amount that is effective to increase the cohesivity of themechanically entangled mass of scaffolding elements. An osteoconductivecalcium-containing particulate material is incorporated in themechanically entangled mass of scaffolding elements in certainbeneficial embodiments. In advantageous forms, an osteoinductive proteincan be included in the composition, and the sulfated glycosaminoglycancan exhibit the capacity to bind the osteoinductive protein. Inaccordance with certain embodiments, the elongate noncollagenousscaffolding elements are ribbons of material having a median width inthe range of about 0.2 mm to about 3 mm, a median length in the range ofabout 5 mm to about 20 mm, and a median thickness in the range of about0.02 mm to about 0.2 mm. Such a composition can be comprised about 60%to about 75% by weight of the liquid organic binder. Also, theosteoinductive protein can be a bone morphogenic protein, for instanceincorporated at a level of about 0.6 milligrams per cubic centimeter(mg/cc) to about 2 mg/cc in the overall composition. Thecalcium-containing particles in the composition can have an averageparticle diameter in the range of about 0.1 millimeters (mm) to about 5mm, and/or can be incorporated at a level of about 0.25 g/cc to about0.35 g/cc in the overall composition. The composition can furtherinclude the scaffolding elements at a level of about 0.04 g/cc to about0.1 g/cc. The viscosity of the liquid organic binder can range fromabout 1 centipoise to about 200 centipoises in certain embodiments. Theosteoinductive composition is desirably a cohesive, shape-retainingputty.

In other embodiments, the invention provides osteoinductive compositionsthat comprise a malleable mechanically entangled mass of elongate,compliant scaffolding elements, wherein said scaffolding elements arecomposed of (i) one or more synthetic polymers, (ii) one or moreplant-derived biopolymers, (iii) one or more insect-derived biopolymers,or (iv) mixtures of two or all of (i), (ii) and (iii). The compositionsinclude a liquid organic binder coating the scaffolding elements,wherein the liquid organic binder has such a viscosity and is present insuch an amount so as to be effective to increase the cohesivity of themechanically entangled mass. An osteoinductive factor is incorporated insaid mechanically entangled mass, and the scaffolding elements exhibit acapacity to bind the osteoinductive factor. In advantageous forms, anosteoinductive protein can be included in the composition, and asulfated glycosaminoglycan can be incorporated in or on the scaffoldingelements and exhibit the capacity to bind the osteoinductive protein. Inaccordance with certain embodiments, the elongate scaffolding elementsare ribbons of material having a median width in the range of about 0.2mm to about 3 mm, a median length in the range of about 5 mm to about 20mm, and a median thickness in the range of about 0.02 mm to about 0.2mm. Such a composition can be comprised about 60% to about 75% by weightof the liquid organic binder. Also, the osteoinductive protein can be abone morphogenic protein, for instance incorporated at a level of about0.6 milligrams per cubic centimeter (mg/cc) to about 2 mg/cc in theoverall composition. The composition can include calcium-containingparticles having an average particle diameter in the range of about 0.1mm to about 5 mm, and/or can be incorporated at a level of about 0.25g/cc to about 0.35 g/cc in the overall composition. The composition canfurther include the scaffolding elements at a level of about 0.04 g/ccto about 0.1 g/cc. The viscosity of the liquid organic binder can rangefrom about 1 centipoise to about 200 centipoises in certain embodiments.The osteoinductive composition is desirably a cohesive, shape-retainingputty.

In other embodiments, provided are methods for preparing anosteoinductive material for inducing bone growth in a patient. Themethods comprise providing an osteoinductive implant material comprisingan osteoconductive scaffolding material incorporating an osteoinductiveprotein. In a sterile operating field, the exterior surfaces of theosteoinductive implant material are coated with a biodegradable barriersubstance to form a coated implant material, wherein the biodegradablebarrier substance is effective to delay release of the osteoinductiveprotein from the scaffolding material.

In other embodiments, provided are methods for inducing bone growth in apatient. The methods comprise providing an osteoconductive scaffoldingmaterial, and incorporating an osteoinductive protein into theosteoinductive scaffolding material to form anosteoinductive-protein-loaded scaffolding material. The method furtherincludes coating exterior surfaces of the loaded scaffolding materialwith a biodegradable barrier substance to form a coated implantmaterial, wherein the biodegradable barrier substance is effective todelay release of the osteoinductive protein from the scaffoldingmaterial. The coated implant material is implanted in a patient at asite at which bone growth is desired.

In another embodiment, provided is an osteoinductive material forimplantation to induce bone growth in a patient. The osteoinductivematerial comprises an osteoinductive implant material including anosteoconductive scaffolding material incorporating an osteoinductiveprotein. A coating is positioned upon exterior surfaces of theosteoinductive implant material, the coating including a biodegradablebarrier substance and being effective to delay release of theosteoinductive protein from the scaffolding material. In certain forms,the scaffolding material exhibits the capacity to bind theosteoinductive protein.

In still further embodiments, the present invention provides methods fortreating patients that involve implanting in the patients at least onemedical composition as described herein, and/or prepared as describedherein.

Additional embodiments as well as features and advantages of the presentinvention will be apparent to those of ordinary skill in the art fromthe descriptions herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a perspective view of a medical product of the inventionincluding a medical paste or putty of the invention packaged within aterminally sterilized syringe device.

FIG. 2 provides a perspective view of a dry disruptable body that can beused to prepare malleable implant compositions of the invention.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to certain embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations and furthermodifications in the illustrated device, and such further applicationsof the principles of the invention as described herein beingcontemplated as would normally occur to one skilled in the art to whichthe invention relates.

As disclosed above, in certain aspects, the present invention relates tomalleable medical implant compositions, including for example putties,to methods and materials that are useful for preparing suchcompositions, and to uses of such compositions. Certain preferredmedical compositions of the invention possess a combination ofadvantageous properties including high mineral content, malleability,cohesiveness, and shape retention. In this regard, as used herein theterm “malleable” means that the material is capable of being permanentlyconverted from a first shape to a second shape by the application ofpressure. The term “cohesive” as used herein to describe a compositionmeans that the composition tends to remain a singular, connected massupon stretching, including the exhibition of the ability to elongatesubstantially without breaking upon stretching. In the context ofstretching compositions of the invention containing insolublebioresorbable ribbons, the advantageous compositions exhibit elongation,during which the existence of substantial levels of intermeshed ribbonsclinging to one another becomes visually apparent. As used herein, theterm “shape-retaining” as used to describe a composition means that thecomposition is highly viscous and unless acted upon with pressure tendsto remain in the shape in which it is placed. This is contrasted tothinner paste form materials which readily flow, and thus would pool orpuddle upon application to a surface in a range of temperatures fromambient room temperature (20° C.) to body temperature (37° C.). Incertain features of the invention, novel combinations of ingredientsprovide a medical material that not only contains a significant, highlevel of large particulate calcium-containing particles, but alsoexhibits superior properties with respect to malleability, cohesiveness,and shape retention.

In certain embodiments, the solid phase of compositions of the inventioncontains elongate scaffolding elements such as fibers, ribbons orstrands that are made from material that is noncollagenous and/or from amaterial composed of synthetic polymers, plant-derived biopolymers,insect-derived biopolymers, or mixtures of some or all thereof. In thisregard, “noncollagenous” as used herein refers to materials that do notcontain any collagen. Scaffolding materials can, for example, beprepared from one or more synthetic polymers. Such polymer(s) can beresorbable. Desirably, resorbable polymers are used and the elongatescaffolding elements will be resorbed within about 2 weeks to about 12weeks after implantation of the composition in a patient, more desirablyabout four to about six weeks. Suitable resorbable synthetic polymersinclude, for instance, aliphatic polyesters, cellulose, copolymers ofglycolide, copolymers of lactide, glycolide/l-lactide copolymers(PGA/PLLA), glycolide/trimethylene carbonate copolymers (PGA/TMC),hydrogel, lactide/tetramethylglycolide copolymers, lactide/trimethylenecarbonate copolymers, lactide/[epsilon]-caprolactone copolymers,lactide/[sigma]-valerolactone copolymers, L-lactide/dl-lactidecopolymers, methyl methacrylate-N-vinyl pyrrolidone copolymers, Nylon-2,PHBA/[gamma]-hydroxyvalerate copolymers (PHBA/HVA), PLA/polyethyleneoxide copolymers, PLA-polyethylene oxide (PELA), poly(amino acids),poly(trimethylene carbonates), poly hydroxyalkanoate polymers (PHA),poly(alklyene oxalates), poly(butylene diglycolate), poly(hydroxybutyrate) (PHB), poly(n-vinyl pyrrolidone), poly(ortho esters),polyalkyl-2-cyanoacrylates, polyanhydrides, polycyanoacrylates,polydepsipeptides, polydihydropyrans, poly-dl-lactide (PDLLA),polyesteramides, polyesters of oxalic acid, polyglycolide (PGA),polyiminocarbonates, polylactides (PLA), poly-l-lactide (PLLA),polyorthoesters, poly-p-dioxanone (PDO), polypeptides, polyphosphazenes,polysaccharides, polyurethanes (PU), polyvinyl alcohol (PVA),poly-[beta]-hydroxypropionate (PHPA), poly-[beta]-hydroxybutyrate (PBA),poly-[sigma]-valerolactone, poly-[beta]-alkanoic acids,poly-[beta]-malic acid (PMLA), poly-[epsilon]-caprolactone (PCL),pseudo-poly(amino acids), trimethylene carbonate (TMC), tyrosine basedpolymers, and others.

Polyhydroxyalkanoate polymers that can be used includepolyhydroxyalkanoate (PHA) polyesters such as polymers or copolymers ofhydroxybutyrates, including 3-hydroxybutyrate and/or 4-hydroxybutyrate.Poly-3-hydroxybutyrate-co-4-hydroxybutyrate (a co-polymer of(R)-3-hydroxybutyrate and 4-hydroxybutyrate) can be used. Other usefulcopolymers in the PHA family includepoly-3-hydroxybutyrate-c-o-3-hydroxyvalerate,poly-hydroxyoctanoate-co-hexanoate andpoly-4-hydoxybutyrate-co-glycolate. Suitable methods for preparing thePHA polyesters are described in Williams, S. F. and Peoples, O. P.CHEMTECH, 26:38-44 (1996), Williams, S. F. and Peoples, O. P., Chem.Br., 33:29-32 (1997), U.S. Pat. No. 4,910,145 to Holmes, P. A. and Lim,G. B.; Byrom, D., Miscellaneous Biomaterials, in D. Byrom, Ed.,Biomaterials MacMillan Publishers, London, 1991, pp. 333-359; Hocking,P. J. and Marchessault, R. H. Biopolyesters, G. J. L. Griffin, Ed.,Chemistry and Technology of Biodegradable Polymers, Chapman and Hall,London, 1994, pp. 48-96; Holmes, P. A., Biologically Produced(R)-3-hydroxyalkanoate Polymers and Copolymers, in D. C. Bassett Ed.,Developments in Crystalline Polymers, Elsevier, London, Vol. 2, 1988,pp. 1-65; Lafferty et al., Microbial Production of Poly-b-hydroxybutyricacid, H. J. Rehm and G. Reed, Eds., Biotechnology, Verlagsgesellschaft,Weinheim, Vol. 66, 1988, pp. 135-176; Muller and Seebach, Angew. Chem.Int. Ed. Engl. 32:477-502 (1993); Steinbuichel, A. PolyhydroxyalkanoicAcids, in D. Byrom Ed., Biomaterials, MacMillan Publishers, London,1991, pp. 123-213; and, Williams and Peoples, CHEMTECH, 26:38-44,(1996); Steinbutchel and Wiese, Appl. Microbiol. Biotechnol., 37:691-697(1992); U.S. Pat. Nos. 5,245,023; 5,250,430; 5,480,794; 5,512,669;5,534,432; Agostini, D. E. et al., Polym. Sci., Part A-1, 9:2775-2787(1971); Gross, R. A. et al., Macromolecules, 21:2657-2668 (1988);Dubois, P. I. et al., Macromolecules, 26:4407-4412 (1993); Le Borgne, A.and Spassky, N., Polymer, 30:2312-2319 (1989); Tanahashi, N. and Doi,Y., Macromolecules, 24:5732-5733 (1991); Hori, Y. M. et al.,Macromolecules, 26:4388-4390 (1993); Kemnitzer, J. E. et al.,Macromolecules, 26:1221-1229 (1993); Hori, Y. M. et al., Macromolecules,26:5533-5534 (1993); Hocking, P. J. and Marchessault, R. H., Polym.Bull., 30:163-170 (1993); Xie, W. et al., Macromolecules, 30:6997-6998(1997), U.S. Pat. No. 5,563,239 to Hubbs, J. C. and Harrison, M. N., andBraunegg, G. et al., J. Biotechnol. 65:127-161 (1998). Suitable PHApolymers can also be obtained commercially from Tepha, Inc. (Cambridge,Mass.) under the trade designations PHA4400 (poly-4-hydroxybutyrate),PHA 3444 (poly-3-hydroxybutyrate-co-4-hydroxybutyrate), and PHA4422.(poly-4-hydoxybutyrate-co-glycolate).

Resorbable insect-derived biopolymers such as resorbable silk materials,for example as available from Serica Technologies, Inc., and/orhyaluronic acid, can also be used to prepare the elongate scaffoldingelement materials used herein. Additionally, resorbable plant-derivedmaterials can be used in the preparation of the elongate scaffoldingelements. Plant-derived materials can include for examplepolysaccharides such as alginates and others identified herein, whichcan be processed to form solid materials by processes includingcrosslinking and/or other techniques. Methods useful for preparing orisolating such polymers or other materials and for forming elongatestrands or other similar scaffolding structures are known in the art andcan be used in accordance with the present invention.

The elongate scaffolding elements can constitute about 2% to about 50%by weight (dry) of the overall composition, more desirably about 10% toabout 50%. It will be understood however that other levels can be usedwithin other aspects of the invention.

In preferred forms, the elongate scaffolding elements will be in theform of ribbons having a median width of at least about 0.2 mm, and incertain forms at least about 0.5 mm. In certain embodiments, the ribbonshave a median width in the range of about 0.2 mm to about 3 mm (morepreferably about 0.5 mm to about 3 mm), a median length in the range ofabout 5 mm to about 20 mm, and a median thickness in the range of about0.02 mm to about 0.2 mm. Such ribbon-form elements can cooperate in thecomposition to provide an effective scaffold for cellular invasion andpenetration through the implanted volume of composition.

In some embodiments, the elongate scaffolding elements will be modifiedto modulate their interaction with one or more morphogenic proteins orother osteoinductive factors. For example, the scaffolding elements canbe modified to provide or enhance their ability to bind to anosteoinductive protein and/or other factor. Illustratively, thescaffolding elements can incorporate and/or be attached to one or moresubstances that promote retention of one or more osteoinductive factors,such as but not limited to morphogenic proteins, in or on the elements.Illustratively, the elongate scaffolding elements can incorporate or beattached to sulfated glycosaminoglycan molecules to which theosteoinductive factor binds. Suitable such sulfated glycosaminoglycansinclude for instance heparin and/or dextran sulfate. The osteoinductivefactor-binding materials may be incorporated into the scaffoldingelements upon their formation and/or pre-formed scaffolding elements canbe attached to (e.g. coated with) the osteoinductive-factor-bindingmaterials. In one mode, heparin or other similar sulfatedglycosaminoglycans can be attached to the surface of the scaffoldingelements by a technique involving end point bonding, e.g. as describedin connection with the Carmeda Bioactive Surface (CBAS) technique inwhich heparin is modified to form an aldehydes group at one end of themolecule, and the aldehyde groups are reacted with the material to becoated with heparin to form an end-point-attachment. Other covalent ornon-covalent modes of bonding the heparin or other osteoinductivefactor-binding ligands to the scaffolding element material or itsprecursor include, for example, ionic bonding, complexation, covalentcrosslinking (e.g. using glutaraldehyde as a coupling agent),thermofixation, and coupling agents such as aminosilane coupling agents.Additionally or alternatively, the surfaces of the scaffolding elementscan be treated to modify their charge, for example adding or enhancing anegative or positive surface charge, in such a fashion as to increasethe ability of the scaffolding elements to bind to a given osteogenicprotein or other factor.

The elongate scaffolding elements can be treated to apply the heparin orother osteoinductive factor-binding coating, and/or the heparin or otherbinding substance may be incorporated within or coated on a precursormaterial that is subsequently processed to form the scaffoldingelements, e.g. by cutting or grinding a precursor solid material,spinning (e.g. melt spinning or electrospinning) or other techniquessuitable for forming scaffolding elements of the modified bioresorbablematerial.

Heparin or another sulfated glycosaminoglycan that is permanently orreleasably bound to the scaffolding elements, and/or other amounts ofheparin or sulfated glycosaminoglycans present in the composition (e.g.an amount dissolved or suspended in the liquid organic binder), can alsoserve to enhance osteoblast differentiation induced by BMPs byprotecting them from degradation and by inhibiting BMP antagonists suchas NOGGIN. The protected BMP can for example be one that is introducedwith the composition and/or one that is natively produced in the patientreceiving the implanted composition.

Malleable compositions of the invention also include a biocompatibleliquid organic binder. In this regard, the term “organic” used in thiscontext means that the liquid binder is or contains at least onecarbon-containing compound. The liquid organic binder can be an aqueousmedium or a non-aqueous medium, and in certain embodiments exhibits aviscosity in the range of 1 centipoise (cps) to 200 cps. The liquidorganic binder can be or comprise one or more of carboxymethylcellulose,glycerin, a polysaccharide such as alginate, or chitosan. In certainaspects, the liquid organic binder will be an aqueous medium comprisinga biocompatible organic substance that is soluble in the medium andincreases the viscosity of the medium. In such embodiments, or otherembodiments, the liquid organic binder can comprise an amount of apolysaccharide compound. In addition, the polysaccharide-containingliquid organic binder, when used, can serve to plasticize the solids inthe composition so as to improve the flow properties and/or reduce thetackiness of the overall malleable composition.

Polysaccharides that can be used as thickeners in the liquid organicbinder include, for example, alginate, hyaluronic acid, chondroitinsulfate, dextran, dextran sulfate, heparin, heparin sulfate, chitosan,gellan gum, xanthan gum, guar gum, and K-carrageenan, or mixtures of twoor more of these or other polysaccharides.

Medical grade polysaccharides suitable for use in aspects of theinvention can be prepared using known techniques or purchased fromcommercial sources. Illustratively, purification techniques forpreparing medical grade polysaccharides may include conventionalseparation techniques such as chromatography, membrane filtration,precipitation, extraction, or other suitable techniques. Medical gradesodium alginate may be commercially obtained, for example, from MedipolSA (Lausanne, Switzerland), or from NovaMatrix FMC Biopolymer(Philadelphia, Pa., Ultrapure PRONOVA brand (endotoxin level<100endotoxin units per gram)).

Alginate polymers contain large variations in the total content of M andG, and the relative content of sequence structures also varies largely(G-blocks, M-blocks and MG alternating sequences) as well as the lengthof the sequences along the polymer chain. In some embodiments, one ormore alginate polymers of the malleable composition can contain morethan 50% alpha-L-guluronic acid. In some embodiments, one or morealginate polymers of the composition can contain more than 60%alpha-L-guluronic acid. In some embodiments, one or more alginatepolymers of the composition can contain 60% to 80% alpha-L-guluronicacid.

In certain embodiments, alginate polymers used in compositions asdescribed herein may have average molecular weights ranging from 2 to1000 kilodaltons (kD). The molecular weight of alginates can affect theproperties of the malleable composition. Generally, lower molecularweight alginates will be more biodegradable. In some embodiments, thealginate polymers have an average molecule weight of from 5 to 350 kD.In some embodiments, the alginate polymers have an average moleculeweight of from 2 to 100 kD. In some embodiments, the alginate polymershave an average molecule weight of from 50 to 500 kD. In someembodiments, the alginate polymers have an average molecule weight offrom 100 to 1000 kD. The molecular weights identified in this paragraphcan similarly apply to other polysaccharides when used in the invention.The alginate, when used, may possess a viscosity in a 1% solutionmeasured at 20 degrees centigrade of from 25 to 1000 mPas and in someembodiments, 50 to 1000 mPas (1% solution, 20° C.), and/or may be usedin a 2% to 20% by weight aqueous solution.

In certain embodiments, the liquid organic binder contains an ionicpolysaccharide that is capable of forming a thermally irreversibleionically-crosslinked gel upon combination with monovalent, divalent orother polyvalent ionic species, in many cases a cationic species.Ionically crosslinkable materials contemplated for use in the practiceof the present invention include ionic materials such as alginates,chitosan, gellan gum, xanthan gum, hyaluronic acid, heparin, pectin,carrageenan, and the like. Many suitable polysaccharides areplant-derived polysaccharides, such as alginates and pectins (includinggel-forming derivatives thereof). Other suitable polysaccharides can bederived from bacteria, including for example gellan gums.

Aqueous solutions of ionic polysaccharides can generally formionically-crosslinked gels upon contact with aqueous solutions ofcounter-ions. For instance, useful agents for ionically crosslinkingalginate, pectin and other similar polysaccharides include cationicgelling agents, preferably including divalent or trivalent cations.Useful divalent cations for these purposes include the alkaline earthmetals, especially calcium and strontium. Aluminum is a usefulcrosslinking trivalent cation. These ionic crosslinking agents willusually be provided by salts. Useful anionic counter-ions for thecalcium or other salts are desirably selected frompharmaceutically-acceptable anions such as chlorides, gluconates,fluorides, citrates, phosphates, tartrates, sulphates, acetates,borates, and the like. An especially preferred ionic crosslinking agentfor use with an alginate or pectin compound is provided by calciumchloride. The ionic polysaccharide chitosan can also be used, and can beionically crosslinked with multivalent, anionic gelling agents. Suchagents include metal polyphosphates, such as an alkali metal or ammoniumpolyphosphate, pyrophosphates or metaphosphates. Citrates can also beused. These anionic crosslinking agents will also usually be provided bysalts. The cationic counter-ion for the polyphosphate or other salt canbe any suitable, biocompatible or pharmaceutically-acceptable cationincluding for instance sodium, potassium, or ammonium. Additionally,polysaccharides which gel by exposure to monovalent cations, includingbacterial polysaccharides, such as gellan gum, and plantpolysaccharides, such as carrageenans, may be crosslinked to form ahydrogel using methods analogous to those available for the crosslinkingof alginates described above. Polysaccharides which gel in the presenceof monovalent cations, such as gellan gums, can also be used. Suchpolysaccharides may form gels upon exposure, for example, to a solutioncomprising physiological levels of sodium. Many other biocompatiblepolysaccharides, including plant-derived and animal-derived materials,as well as corresponding ionic crosslinking agents, are known and canalso be used in aspects of the present invention.

When ionic polysaccharides that are capable of formingthermally-irreversible ionically-crosslinked gels are used in the liquidorganic binder, it will be understood that in malleable compositions ofthe present invention these polysaccharides will be ionicallycrosslinked, if at all, only to an extent that does not eliminate themalleable nature of the implant material. Thus, within aspects of thepresent invention, no or substantially no ionic crosslinking agent willbe added, or in some cases only a relatively small amount of ioniccrosslinking agent can be added in order to increase the viscosity ofthe overall formulation. On the other hand, in other aspects of theinvention, malleable compositions as described herein can be contactedwith an amount of a liquid medium containing an ionic crosslinking agentimmediately prior to, during, or after implantation of the material intoa patient. Illustratively, a malleable composition as described hereincan be co-administered with a liquid medium of ionic crosslinker, as inthe case of a dual-barrel syringe administration by which the malleablecomposition and crosslinker are admixed as they exit the syringe.Alternatively, a previously-implanted amount of the malleablecomposition can be washed with a solution or other liquid mediumcontaining an appropriate ionic crosslinking agent to stiffen theimplanted material in situ.

In certain embodiments, compositions of the invention include theinsoluble elongate scaffolding elements at a level of 0.04 g/cc to 0.1g/cc of the composition, and/or include a polysaccharide (e.g. in theliquid organic binder) at a level of 0.01 g/cc to 0.08 g/cc of thecomposition. In other embodiments, such compositions include theinsoluble bioresorbable scaffolding elements at a level of about 0.05 to0.08 g/cc in the composition. When one or more polysaccharides are usedin an aqueous binder, advantageous compositions can include theinsoluble elongate scaffolding elements in an amount (percent by weight)that is at least equal to or greater than the total amount ofpolysaccharide(s), to contribute beneficially to the desired handlingand implant properties of the compositions, e.g. in the provision of ashape-retaining putty material.

In certain forms, medical putties or other compositions of the presentinvention also include an amount of a particulate calcium-containingmaterial. In certain aspects of the invention, such a particulatematerial is incorporated in the inventive composition at a level of atleast about 0.25 g/cc of composition, and typically in the range ofabout 0.25 g/cc to about 0.35 g/cc. Such relatively high levels ofcalcium-containing particles can be helpful in providing additionalscaffold material for the ingrowth of new bone.

The calcium-containing particulate material used in the presentinvention can include a natural or synthetic mineral or mixture ofmineral materials that is effective to provide a scaffold for boneingrowth. Illustratively, a mineral matrix may be selected from one ormore materials from the group consisting of bone particles, Bioglass®,tricalcium phosphate, biphasic calcium phosphate, hydroxyapatite,corraline hydroxyapatite, and biocompatible ceramics. Biphasic calciumphosphate is a particularly desirable synthetic ceramic for use in theinvention. Such biphasic calcium phosphate can have a tricalciumphosphate:hydroxyapatite weight ratio of about 50:50 to about 95:5, morepreferably about 70:30 to about 95:5, even more preferably about 80:20to about 90:10, and most preferably about 85:15. The calcium-containingparticulate material can have an average particle diameter between about0.1 and 5 mm, more typically between about 0.1 and 2 mm.

In certain embodiments, the calcium-containing particulate incorporatedinto the inventive composition can include a substantial component ofrelatively larger particles in combination with relatively smallerparticles. In certain aspects, the calcium-containing particulate can beconstituted at least 10 weight % by particles having a maximum dimensionof greater than about 2 mm, or greater than about 3 mm (e g in the rangeof about 3 mm to about 5 mm) and at least 10 weight % by particleshaving a maximum dimension of less than about 1 mm. In further aspects,the calcium-containing particulate can be constituted at least 20 weight% by particles having a maximum dimension of greater than about 2 mm, orgreater than about 3 mm (e g in the range of about 3 mm to about 5 mm)and at least 20 weight % by particles having a maximum dimension of lessthan about 1 mm. In still further embodiments, the calcium-containingparticulate can be constituted about 10 weight % to about 40 weight % byparticles having a maximum dimension of greater than about 2 mm, orgreater than about 3 mm (e g in the range of about 3 mm to about 5 mm)and about 90 weight % to about 60 weight % by particles having a maximumdimension of less than about 1 mm. It will be understood that particlesas described above may have the given dimensions along one axis or alongtwo or three axes. Products having such calcium-containing particlesizes can be prepared, for example, by blending separate particulateproducts having the respective particle size distributions. The presenceof relatively large calcium-containing particles in combination withsmaller particles can provide an overall composition that resistscompression upon impingement by soft tissues at an implant site, andthat also can exhibit beneficial handling and implant properties.

In certain embodiments, the calcium-containing particulate materialconstitutes about 50% to about 98% by weight (dry) of the overallcomposition, more desirably about 70% to about 90%. It will beunderstood however that other levels of such particulate material can beused in other aspects of the invention.

Malleable compositions of the invention can include a significantproportion of the liquid organic binder, for instance with the liquidorganic binder constituting about 50% or more by weight of the overallcomposition. In one aspect, a malleable, cohesive, shape-retainingcomposition of the invention comprises about 60% to about 75% by weightof the liquid organic binder material. It will be understood howeverthat higher or lower levels of the liquid binder can also be used toprepare either firmer (e.g. dampened solid matrices) or more flowablematerials, such as pastes, for implantation in a patient. As will alsobe appreciated by those skilled in the art, in many instances, the pH,ionic strength, and/or other similar characteristics of the liquidbinder can be selected to control its viscosity. Illustratively, theseand other parameters of the liquid binder component can be controlled toprevent undesired levels of ionic crosslinking of any polysaccharide(s)present that would unduly disrupt the malleable character of a preferredimplant composition.

In certain embodiments, the osteoinductive substance can include one ormore growth factors that is/are effective in inducing formation of bone.Desirably, the growth factor will be from a class of proteins knowngenerally as bone morphogenic proteins (BMPs), and can in certainembodiments be recombinant human (rh) BMPs. These BMP proteins, whichare known to have osteogenic, chondrogenic and other growth anddifferentiation activities, include rhBMP-2, rhBMP-3, rhBMP4 (alsoreferred to as rhBMP-2B), rhBMP-5, rhBMP-6, rhBMP-7 (rhOP-1), rhBMP-8,rhBMP-9, rhBMP-12, rhBMP-13, rhBMP-15, rhBMP-16, rhBMP-17, rhBMP-18,rhGDF-1, rhGDF-3, rhGDF-5, rhGDF-6, rhGDF-7, rhGDF-8, rhGDF-9, rhGDF-10,rhGDF-11, rhGDF-12, rhGDF-14. For example, BMP-2, BMP-3, BMP-4, BMP-5,BMP-6 and BMP-7, disclosed in U.S. Pat. Nos. 5,108,922; 5,013,649;5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCTpublication WO91/18098; and BMP-9, disclosed in PCT publicationWO93/00432, BMP-10, disclosed in U.S. Pat. No. 5,637,480; BMP-11,disclosed in U.S. Pat. No. 5,639,638, or BMP-12 or BMP-13, disclosed inU.S. Pat. No. 5,658,882, BMP-15, disclosed U.S. Pat. No. 5,635,372 andBMP-16, disclosed in U.S. Pat. Nos. 5,965,403 and 6,331,612. Othercompositions which may also be useful include Vgr-2, and any of thegrowth and differentiation factors including those described in PCTapplications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681;WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others. Also usefulin the present invention may be BIP, disclosed in WO94/01557; HP00269,disclosed in JP Publication number: 7-250688; and MP52, disclosed in PCTapplication WO93/16099. The growth factor may also be a LIMMineralization Protein (LMP), including for example one or more of thosedisclosed in U.S. Pat. Nos. 6,858,431 and 7,045,614. The disclosures ofall of these patents and applications are hereby incorporated herein byreference. Also useful in the present invention are heterodimers of theabove and modified proteins or partial deletion products (biologicallyactive fragments) thereof. These proteins can be used individually or inmixtures of two or more. rhBMP-2 or biologically active derivatives orfragments thereof that exhibit an ability to induce bone growth arepreferred.

The BMP or other osteoinductive protein may be recombinantly produced.The BMP may be homodimeric, or may be heterodimeric with other BMPs(e.g., a heterodimer composed of one monomer each of BMP-2 and BMP-6) orwith other members of the TGF-beta superfamily, such as activins,inhibins and TGF-beta 1 (e.g., a heterodimer composed of one monomereach of a BMP and a related member of the TGF-beta superfamily).Examples of such heterodimeric proteins are described for example inPublished PCT Patent Application WO 93/09229, the specification of whichis hereby incorporated herein by reference. The amount of osteogenicprotein useful herein is that amount effective to stimulate increasedosteogenic activity of infiltrating progenitor cells, and will dependupon several factors including the size, location, and nature of thedefect being treated, and the carrier and particular protein beingemployed. In certain embodiments, the amount of osteogenic protein to bedelivered to the implant site will be in a range of from about 0.05 toabout 1.5 mg.

Compositions described herein can include a bone morphogenic protein oranother osteoinductive factor incorporated therein in an effectiveamount to render the composition osteoinductive when implanted in amammal, such as a human patient. In one embodiment, an inventivecomposition includes a bone morphogenic protein or other osteogenicprotein at a level of about 0.6 milligrams per cubic centimeter (mg/cc)of putty to about 2 mg/cc of putty, advantageously at a level of about0.8 mg/cc to about 1.8 mg/cc.

Other therapeutic growth factors or substances may also be used inputties or other compositions of the present invention, especially thosethat can be used to stimulate bone formation. Such proteins are knownand include, for example, demineralized bone matrix, platelet-derivedgrowth factors, insulin-like growth factors, cartilage-derivedmorphogenic proteins, statins, and transforming growth factors,including TGF-α and TGF-β.

The osteoinductive proteins and/or other biologically active agents tobe used in the present invention can be provided in liquid formulations,for example buffered aqueous formulations. In certain embodiments, suchformulations are mixed with, received upon and/or within, or otherwisecombined with a dried implant material which is then manipulated toprepare a malleable osteoinductive material of the invention.

As further enhancements of the compositions of the present invention,those skilled in the art will readily appreciate that other biologicallyactive agents can be incorporated into the compositions. Such additionalagents include, but are not limited to, microglobulin-beta, antibiotics,antifungal agents, wetting agents, steroids and non-steroidalanti-inflammatory compounds.

In another aspect, the present invention provides a dry implant materialthat can be combined with an appropriate amount of a liquid medium inorder to prepare a malleable implant material as described herein. Thedry implant material can be in any suitable form, including for examplea mixed powder. In advantageous forms, the dry implant material will bea porous body that includes the bioresorbable scaffolding elementmaterial. The body can also include a particulate calcium-containingmaterial as described herein embedded within the disruptable matrix. Thedried, porous implant body or other dry material can be comprised about70% to about 90% by weight of the particulate calcium-containingmaterial and about 10% to about 30% by weight of the scaffolding elementmaterial. When a water-soluble organic compound, for example apolysaccharide, is to be incorporated in a liquid organic binder, it canalso be incorporated in the dried porous body or other material, e.g. inan amount of about 1% to about 15% by weight. In certain embodiments,the dried, porous implant body or other material can include apolysaccharide in an amount from about 6% to about 10% by weight. Inalternative embodiments that ultimately use a polysaccharide(s) or otherorganic substances in the liquid organic binder, the dried material canbe free of the polysaccharide or other organic substance, but thewetting medium applied to dried material can include the polysaccharideor other organic substance. Combinations of these features are alsocontemplated herein, e.g. wherein one or more polysaccharides or otherbinder-forming organic substances, are included in each of the wettingmedium and the dried material. The organic binder-forming substances inthe wetting medium and dried material can also be the same as, ordifferent from, each other. These and other alternatives will beapparent to those of ordinary skill in the art from the descriptionsherein.

In certain embodiments, a dried, porous body as discussed above can havea density of between about 0.1 g/cc to about 0.3 g/cc, more desirablybetween about 0.15 g/cc and about 0.25 g/cc. Such dried, porous implantbodies can also exhibit porosities of at least about 50%, more desirablyat least about 70% up to about 90%, and in certain embodiments in therange of about 80% to about 90%.

Dried, porous implant bodies in accordance with the invention can beprovided in any suitable shape, including cylinders, cubes, chunks,pellets or other shapes. In certain aspects, the dried, porous implantbody can define a reservoir for receiving amounts of a wetting liquid,e.g. to be used in the preparation of a putty or other malleablematerial from the dried implant material or used simply to wet the bodyfor implant.

The dried porous body can be prepared using any suitable technique,including for example casting a liquid medium containing the dryingredients, and then drying that medium by any appropriate means suchas air drying or lyophilization. In this regard, the concentration ofsolids and other material in the liquid medium can be adjusted tocontrol the ultimate porosity of the dried material, with lower solidsconcentrations typically providing higher porosities, and higher solidsconcentrations typically providing lower porosities. Additionalvariation of the porosity may be imparted by modifying the dryingtechnique, including for example my modifying the lyophilization cycleto avoid substantial collapse or contraction of the cast material duringdrying. These and other porosity control parameters may be manipulatedto control the porosity of the formed body.

Compositions of the present invention can be manufactured in aready-to-use format and packaged in a medically acceptable container forwetted malleable materials. In some embodiments, as illustrated in FIG.1, the ready-to-use medical product can be a product 11 including asyringe device 12 containing an amount of a malleable composition 13 ofthe invention. The composition is contained within syringe barrel 14,and is transferable from the barrel 14 by actuating a plunger 15.

Compositions of the invention can also be prepared on-site, at or nearthe time of surgery. For instance, dry materials can be provided in theappropriate amounts in a dried porous body as discussed above, and asuitable liquid such as a buffered aqueous solution or a liquid organicbinder as described above can be combined with the dry materials andmixed to form malleable implant compositions. With reference to FIG. 2,in one form, an inventive product 21 includes a body 22 of dry materialsincluding at least the elongate scaffolding material (e.g. includingbound heparin or other sulfated glycosaminoglycans) and acalcium-containing particulate material is provided. The body 22 of drymaterials may also include an organic binder material (e.g. one or morepolysaccharides) and/or an osteogenic factor. Body 22 can optionallydefine a reservoir 23 for receiving and retaining amounts of the liquidmedium to be combined with the dry materials as it soaks into the body22. The body 22 can thereafter be disrupted, e.g. by manual kneading,mixing or otherwise, to form a malleable composition of the invention.

Typically, the combination of a disruptable, dried porous implant bodyof the invention with a liquid, and the physical kneading or othermixing of the resultant mass, will result in a reduction of the volumeof the dried porous body, for example resulting in a malleablecomposition volume that is about 30% to about 70% of that of theoriginal implant body, more typically about 40% to about 60%. This is aresult of a breakdown of the original porosity of the dried implant bodyto form a relatively less porous or non-porous malleable implantcomposition. The liquid can be an aqueous substance, including forinstance sterile water, physiological saline or other solutions (with orwithout organic co-solvents), emulsions or suspensions that provideadequate wetting characteristics to form malleable compositions of theinvention.

In use, the implant compositions of the invention can be implanted at asite at which bone growth is desired, e.g. to treat a disease, defect orlocation of trauma, and/or to promote artificial arthrodesis. Themalleable form of certain inventive compositions enables theirpositioning, shaping and/or molding within voids, defects or other areasin which new bone growth is desired. In particularly advantageousembodiments, the malleable composition will be in the form of ashape-retaining putty material that provides sufficientthree-dimensional integrity to resist substantial compression whenimpinged by adjacent soft tissues of the body at a bony implant site.

In certain embodiments, all insoluble solids in compositions of theinvention will be free from collagen. In other embodiments, the entirecompositions of the invention will be free from collagen. In furtherembodiments, the insoluble solids in compositions of the invention willbe free or essentially free from any mammalian-derived protein, oralternatively free or essentially free from any animal-derived protein.In still further embodiments, the entire compositions of the inventionwill be free or essentially free from mammalian-derived protein, oralternatively free or essentially free from any animal-derived protein.The term “essentially free from” as used in this context excludes thepresence of the identified substance (e.g. collagen or protein material)other than that which might occur as a trace impurity, e.g. in thepreparation of other materials (e.g. non-collagen materials ornon-protein materials) derived from mammalian or other animal tissue.

Bone repair sites that can be treated with compositions of the inventioninclude, for instance, those resulting from injury, defects broughtabout during the course of surgery, infection, malignancy ordevelopmental malformation. The compositions can be used in a widevariety of orthopedic, periodontal, neurosurgical and oral andmaxillofacial surgical procedures including, but not limited to: therepair of simple and compound fractures and non-unions; external andinternal fixations; joint reconstructions such as arthrodesis; generalarthroplasty; cup arthroplasty of the hip; femoral and humeral headreplacement; femoral head surface replacement and total jointreplacement; repairs of the vertebral column including spinal fusion andinternal fixation; tumor surgery, e.g., deficit filing; discectomy;laminectomy; excision of spinal cord tumors; anterior cervical andthoracic operations; repairs of spinal injuries; scoliosis, lordosis andkyphosis treatments; intermaxillary fixation of fractures; mentoplasty;temporomandibular joint replacement; alveolar ridge augmentation andreconstruction; inlay osteoimplants; implant placement and revision;sinus lifts; cosmetic enhancement; etc. Specific bones which can berepaired or replaced with the compositions include, but are not limitedto: the ethmoid; frontal; nasal; occipital; parietal; temporal;mandible; maxilla; zygomatic; cervical vertebra; thoracic vertebra;lumbar vertebra; sacrum; rib; sternum; clavicle; scapula; humerus;radius; ulna; carpal bones; metacarpal bones; phalanges; ilium; ischium;pubis; femur; tibia; fibula; patella; calcaneus; tarsal and metatarsalbones.

Where a composition of the invention is osteoinductive, once in place,it can effectively induce the ingrowth of bone into the desired areaeven in a human or other primate subject such as a human exhibiting arelatively slow rate of bone formation compared to smaller mammals, forexample rodents or rabbits.

Compositions of the invention are especially advantageous when used inbones or bone portions that are vascularized to only moderate or lowlevels. These areas present particularly low rates of bone formation,and as such the rapid resorption of the carrier poses enhanceddifficulties. Examples of moderate or only slightly vascularized sitesinclude, for example, transverse processes or other posterior elementsof the spine, the diaphysis of long bones, in particular the middiaphysis of the tibia, and cranial defects. An especially preferred useof compositions of the invention is as an implant to promote arthrodesisbetween vertebrae in spinal fusions in humans or other mammals,including for example interbody, posterior and/or posterolateral fusiontechniques.

In addition, in accordance with other aspects of the invention, thecompositions of the invention can be incorporated in, on or around aload-bearing spinal or other orthopedic implant device (e.g. having acompressive strength of at least about 10000 N) such as a fusion cage,dowel, or other device having a pocket, chamber or other cavity forcontaining an osteoinductive and/or osteoconductive composition, andused in a spinal fusion such as an interbody fusion. In one illustrativeexample, an inventive composition can be used in conjunction with aload-bearing interbody spinal spacer to achieve an interbody fusion.

In some embodiments, medical compositions of the present invention canbe used in combination with cells, including for instance progenitorand/or stem cells derived from embryonic or adult tissue sources and/ortaken from culture. Illustratively, compositions of the invention canincorporate cells derived from blood, bone marrow, or other tissuesources from the patient to be treated (autologous cells) or from asuitable allogenic or xenogenic donor source. In certain embodiments ofthe invention, putties of the invention incorporate an enriched bonemarrow fraction, prepared for example as described in US PatentPublication No. 2005/0130301 to McKay et al. published Jun. 16, 2005,publishing U.S. patent application Ser. No. 10/887,275 filed Jul. 8,2004, which is hereby incorporated herein by reference in its entirety.Thus, the implant materials can incorporate a bone marrow fractionenriched in connective tissue growth components, that is prepared bycentrifuging a biological sample (e.g. from the patient to be treated)to separate the sample into fractions including a fraction rich inconnective tissue growth components. The fraction rich in connectivetissue growth components can then be isolated from the separated sample,and incorporated into the putty material of the present invention, e.g.by using the fraction in or as the wetting medium for the dried, porousbody as discussed hereinabove.

The present invention also provides medical kits that can be used toprepare implant compositions. Such kits can include a dried material(e.g. a dried porous body) as described herein, along with a liquid(e.g. aqueous) medium for combination with the body to form malleableimplant composition or another wetted implant form, and/or another kititem such as a load-bearing implant (e.g. a spinal spacer) and/or anosteoinductive substance such as a BMP. In one specific form, such amedical kit will include the dried, porous body, a BMP in lyophilizedform (e.g. rhBMP-2), and an aqueous medium for reconstitution of the BMPto prepare an aqueous formulation that can then be combined with thedried, porous body to prepare an osteoinductive putty or other wettedimplant form of the invention.

In certain aspects of the invention, the liquid organic binder material,or another suitable biocompatible material, may be used as a barriermaterial in such a manner as to function as a physical barrier to delaythe release of an osteogenic factor, such as an osteogenic protein, fromthe scaffolding material. In this regard, the osteogenic protein orother active factor can first be incorporated into or onto thescaffolding material, and the scaffolding material can then be coatedwith the barrier material sufficiently to delay the release of theprotein or other factor from the scaffolding material. In preferredembodiment, the scaffolding material is transferred to the sterilepatient operating field, and the barrier material is applied in thesterile operating field. Thus, in certain forms, a scaffolding materialsuch as that described herein can be removed from a sterile package inthe sterile operating field, and the medical care provider can thenapply the barrier material as a coating upon the scaffolding materialwhile in the operating field. The osteogenic protein or other factor canalso be applied to the scaffolding material while in the sterileoperating field, for example prior to the application of the barriermaterial. Alternatively or in addition, the osteogenic protein or otherfactor can be applied to the scaffolding material prior to entry intothe sterile operating field, for example during product manufacture(e.g. as in the case where the sterile package contains the scaffoldingmaterial with a pre-applied amount of the osteogenic protein or otherfactor). In some modes of practice, the scaffolding material may exhibitthe capacity to bind the osteogenic protein or other factor, e.g. byitself or with the addition of another material such as a sulfatedglycosaminoglycan as described herein. In this manner, both a binding ofthe protein or other factor to the scaffolding material and the physicalbarrier material can beneficially delay release of the protein or otherfactor from the scaffolding material. This delay in release canbeneficially maintain the protein or other factor within the implantedvolume of scaffolding material for a longer period of time, thusproviding an improved generation of bone through the implanted volume ofscaffolding material. This delay in release can also be beneficial ingenerating bone formation through the implanted volume with less proteinas compared with an equivalent scaffold without delayed release ofprotein.

The applied barrier material as discussed above can be a bioabsorbablesubstance. In this fashion, the barrier material can function as atemporary barrier to the release of the osteogenic factor, andthereafter become absorbed by the body of the patient. The barriermaterial can be selected and applied such that it is absorbed within arelatively short period of time after implant, for example within abouttwo weeks. In certain forms, the barrier material will be substantiallyabsorbed within about three to about seven days. In this fashion, thebarrier material can promote early retention of the osteoinductivefactor at the implant site during a period soon after implant in whichnative healing responses flush the implant site with bodily fluids andin which a native blood clot is formed in and around the implant region.After formation of the clot, the barrier material will have served itsfunction of resisting early wash-out of the osteoinductive factor fromthe implanted composition and can be absorbed by the body soonthereafter.

In malleable compositions such as those described hereinabove, thebarrier material can also serve as a binder, and can be incorporatedsubstantially homogenously throughout the composition. In otherembodiments, a coating or layer of barrier material can be selectivelyapplied to an external surface of a volume of an implant compositionsuch that it is non-homogenously incorporated in the overall implantmaterial. In such embodiments, the volume of implant composition that iscoated with barrier material can comprise a malleable composition and/ora three-dimensionally stable composition such as a sponge or otherporous matrix. The implant composition can be loaded with the osteogenicfactor (e.g. in the sterile field or during manufacture) and thenexternally coated with the barrier material prior to implantation withinthe patient.

The invention will now be more particularly described with reference tothe following specific Examples. It will be understood that theseExamples are illustrative and not limiting of the invention.

Example 1 Preparation of Dried Implant Body

Sodium alginate is added with mixing to a preparation containingnoncollagenous fibers (e.g. selected from among those listed below)adjusted with NaOH to have a pH in the range of 6.5-7.0. To this mixtureare added biphasic calcium phosphate ceramic granules, with mixing. Adried, porous body is prepared by casting the resulting mixture into acylindrical form and then lyophilizing the cast mixture. The relativeamounts of the constituents of the dried body are as follows:

Material Wt % Solids Biphasic CaP Granules* 75% Insoluble NoncollagenousFibers (resorbable silk, 15%poly-3-hydroxybutyrate-co-4-hydroxybutyrate, or other materialsidentified herein) Sodium Alginate 10% *Mastergraft ® Ceramic Granules,biphasic calcium phosphate granules containing 85% tricalcium phosphateand 15% hydroxyapatite, particle size 0.5-1.6 mm.

Example 2 Preparation of Osteoconductive Putty

Phosphate buffered saline (PBS) is added to the body of Example 1, andthe body is disrupted and kneaded to form a malleable putty. The PBS isadded in sufficient amount that water constitutes about 70% by weight ofthe formed putty. The putty formed is a malleable, cohesive, fibrousmass with entrained ceramic granules.

Example 3 Preparation of Osteogenic Putty

A buffered aqueous solution of rhBMP-2 (1.5 mg/ml solution, as availablewith INFUSE® Bone Graft, Medtronic Sofamor Danek, Memphis, Tenn.) isadded to the body of Example 1, and the body is disrupted and kneaded toform a malleable putty. The rhBMP-2 solution is added in sufficientamount that water constitutes about 70% by weight of the formed putty.The putty formed is an osteogenic, malleable, cohesive, fibrous masswith entrained ceramic granules.

Example 4 Use of Inventive Putty in Interbody Spinal Fusions

An ovine interbody fusion model is used to compare the ability of a 5mm×11 mm×11 mm polyetheretherketone spinal spacer (VERTE-STACK®CORNERSTONE® PSR PEEK Implant, Medtronic Sofamor Danek, Memphis, Tenn.)with packed-in Autograft and the PEEK spacer with packed-in OsteogenicPutty of Example 3 to effect interbody fusion at 6 monthspost-operatively. The efficacy of these treatments to induce interbodyfusion in the ovine lumbar fusion model using blinded radiographic,biomechanical, and histologic measures is evaluated. Assessment offusion is made with Faxitron high-resolution radiography,non-destructive biomechanical testing, and undecalcified histology withcorresponding microradiography. All analyses are conducted in a blindedfashion. In addition, undecalcified histology is used to evaluate theosteocompatibility of the Example 3 Putty. In addition to the treatmentgroups being evaluated, normal spines are evaluated using the samemethodology. When all data acquisition is complete, the key is broken,and radiographic, biomechanical, and histologic data are analyzed bytreatment group.

Animal Model:

The sheep lumbar spine model is used because of the biomechanicalsimilarities between the sheep and human lumbar spine. Wilke et al.characterized the biomechanical parameters (range of motion, neutralzone, and level stiffness) of sheep spines and made comparisons withdata from human specimens previously published by White and Panjabi(see, Wilke et al., Spine: 22(20): 2365-2374, 1997; and White A A andPanjabi M M, editors, Clinical Biomechanics of the Spine, 2nd ed., J. B.Lippincott, Philadelphia, Pa., 1990). Wilke et al. found that the“ranges of motion of sheep spines for the different load directions arequalitatively similar in their craniocaudal trends to those of humanspecimens reported in the literature.” They further concluded that:“Based on the biomechanical similarities of the sheep and human spinesdemonstrated in this study, it appears that the sheep spine . . . canserve as an alternative for the evaluation of spinal implants.

Surgical Technique:

Upon arrival at the facility, the 12 sheep are placed in the appropriatepastures of the large animal research barn. They are dewormed andeartagged for identification. Physical examination is performed and anyanimals with signs of respiratory disease had venous blood submitted fora complete blood count (CBC).

The sheep are anesthetized. Wool is removed from the dorsal lumbar areaand the sheep positioned in sternal recumbency on the operating table.

Iliac Crest Autograft Harvesting:

Autograft is used as a control. The following protocol is followed. Thedorsal and dorsolateral lumbar and iliac crest areas are prepared foraseptic surgery with multiple scrubs of povidone-iodine alternated withisopropyl alcohol. The area is draped and a 3-cm incision made over theleft iliac crest. Following partial reflection of the gluteal muscles,an osteotome is used to create a small window in the craniodorsal faceof the iliac crest. Using a curette, about 2 cc of autogenous cancellousbone is removed, and is later packed into one of the implants (e.g. PEEKspacer) used for the lumbar fusion (this is the control case).Intralesional morphine sulfate is administered prior to closure of theiliac crest incision. The iliac crest site is closed routinely using 2/0polysorb for the subcutaneous tissues and stainless steel staples forthe skin.

Ventral (“Anterior”) Interbody Fusion:

The dorsal and dorsolateral lumbar area is prepared for aseptic surgerywith multiple scrubs of povidone-iodine alternated with isopropylalcohol. The area is draped and a ventrolateral retroperitoneal approachto L3/L4 and L5/L6 through the oblique abdominal muscles to the planeventral to the transverse processes is made.

Implant Insertion:

The bone graft from the iliac crest or the Example 3 Putty is placedinto the PEEK spacer (˜1.5 cc of material) and implanted into the discspace, following preparation of the endplates.

Wound Closure:

Routine closure of external abdominal muscular fascia (0 Polysorb(absorbable suture), subcutaneous tissue (2/0 Polysorb and skin (2/0monofilament non-absorbable suture) is performed. Operative time foreach animal is about 40 minutes. Perioperative antibiotics (Cephazolinsodium) are administered. Postoperative radiographs are performed whilethe sheep are still under general anesthesia.

Aftercare:

Immediately after surgery, the sheep are transferred from the operatingtable to a modified wheelbarrow and while still under generalanesthesia, taken to a radiology suite where dorsoventral and lateralradiographs of the fusion sites are obtained. Following radiographicevaluation, while still in the modified wheelbarrow, they are observeduntil the swallowing reflex returns. At that point they are extubatedand taken to a trailer where they are propped in sternal recumbency. Atthe end of the day, all animals that are operated upon that day aremoved to research pastures. The sheep are housed outdoors (with accessto a three-sided shelter) for the convalescence and allowed to exerciseat will. Postoperative analgesia is provided as described. The sheep areanesthetized and radiographed at three months posoperatively.

Euthanasia:

After 6 months postoperatively, the 12 sheep are euthanized in a humanemanner. Euthanasia is performed according to the guidelines set forth bythe AVMA Panel on Euthanasia (J. Am. Vet. Med. Assoc., 202:229-249,1993). Radiographs of the lumbar fusion sire are taken in these sheep toevaluate the degree of fusion at L3-L4.

Specimen Collection and Handling:

Following euthanasia, a complete gross necropsy is conducted on all 12animals. Conventional gross examination of all major organ systems andhistopathological evaluation of any pathological lesions is performed.Any animals that die or are prematurely euthanized during the course ofthe study have a complete necropsy performed to determine the cause ofdisease or death. At necropsy the lumbar vertebrae that are fused areharvested.

Material Analysis:

All samples from the lumbar area from the sheep are subjected tomechanical testing of the fusion sites. They are tested for stiffness tosaggital and coronal plan bending moments (flexion, extension, right andleft lateral bending). As these mechanical tests are nondestructive, thefusion sites are also examined histologically.

Implant Materials:

Twelve treated spinal levels (L4-L5) are evaluated. The study groups aredefined below.

Study Group No. of Samples (N) Autograft Interbody w/PEEK spacer 6(Autograft + PEEK) Example 3 Putty w/PEEK spacer 6 Normal Intact 17Total 29After the survival phase of the study is completed, the spines areimmediately frozen for evaluation.Methods of Analysis:1. Ex-Vivo Biomechanical Testing of the Treated Lumbar Motion Segment:Flexibility Testing

Unconstrained biomechanical testing is performed in a non-destructivemanner on all spines after the frozen specimens are thawed. All testsare performed within 12 hours of specimen thawing. Specimens are onlyfrozen once. Instrumentation applied to the anterior part of thevertebral body is removed prior to biomechanical testing so that onlythe stiffness of the spine and fusion mass construct is tested, not theinstrumentation. Flexibility of the motion segments is determined inflexion, extension, right and left lateral bending, and right and leftaxial rotation. The purpose of the biomechanical testing is to quantifythe stiffness of the lumbar motion segments augmented with thepreviously described interbody fusion treatments. The treated (L4-L5)motion segments are dissected from the harvested lumbar spines andcleaned of extraneous soft tissues leaving the ligamentous and osseoustissues intact. Specially designed loading and base frames are securedon the L4 and L5 vertebra, respectively.

Moments of 0, 0.5, 2.5, 4.5, 6.5, and 8.5 Nm are achieved in eachloading direction. Static loads are used to apply the pure moments.Three markers reflecting the infrared light are attached to eachvertebra. The locations of the infrared reflective markers are recordedusing three VICON cameras (ViconPeak, Oxford, England) at each load.Three-dimensional load-displacement data are then acquired with puremoments applied in flexion, extension, left and right lateral bending,and left and right axial rotation. Basic principles of using 3-D motionanalysis system for investigating the 3-D load-displacement behavior arewell known in the literature.

Biomechanics data from a normal (untreated) intact group of sheep lumbarspine motion segments that have been obtained previously are used asbaseline data for normal lumbar spine motion for L4-L5 in sheep.Differences in the stiffness (flexibility) between groups and thenormals are statistically compared. Non-parametric Kruskal-Wallis andMann-Whitney tests are used to analyze the biomechanics data.

2. Radiographic Assessment:

Radiographs are taken immediately after surgery (AP and lateral views),at regular post-operative intervals (AP and lateral views), and at thetime of sacrifice (AP and lateral views). A high-resolution radiographyunit (Faxitron, Hewlett Packard, McMinnville, Oreg.) and high-resolutionfilm (EKTASCAN B/RA Film 4153, Kodak, Rochester, N.Y.) are used toproduce a high-resolution PA and lateral radiograph of the harvestedlumbar spines after biomechanical testing. Radiographs are scanned usingimage analysis software (Image Pro Plus Software v 5.0, MediaCybernetics, Silver Spring, Md.) running on a Windows XP workstation. Avideo camera (Model DFC 280, Leica Microsystems, Cambridge, UK) is usedto acquire the digital images of the radiographs. These radiographs arealso used to gross the samples for histologic analyses as outlinedbelow.

Three blinded evaluators evaluate the resulting Faxitron radiographs forinterbody fusion. On the lateral radiographs, the center of the discspace as well as the anterior and posterior margins are evaluated forfusion based on the following scoring method: 4=continuous bonybridging, 3=increased bone density, 2=lucency with some bony bridging,and 1=non-fusion. Lastly, based on both the P/A and lateral radiographs,the blinded evaluators rate an overall fusion score for the spinal levelusing the following criteria:

-   -   3=Solid interbody fusion with no radiolucencies in interbody        space    -   2=Probable fusion with radiolucencies in the interbody space    -   1=Non-fusion with significant radiolucencies in the disc space        with no evidence of superior to inferior bony bridging        3. Undecalcified Histology and Microradiography:

Processing and Stained Undecalcified Sections

In all of the treatment groups, the bisected spinal level is analyzedusing undecalcified techniques (microradiographs and multiple stain).Differential staining along with qualitative optical microscopy isperformed to assess bony bridging and extent of fusion associated withthe autograft or the bone graft substitute packed within the PEEKspacers. Differential staining is used to evaluate the extent of fusionadjacent to and within the PEEK spacers, the host response to the PEEKspacer and Example 3 Putty (if present), the interface of the PEEKspacer, bone graft and Example 3 Putty incorporation, and boneremodeling within the fusion mass.

After Faxitron radiography, all spinal levels containing an implant aregrossed in the following manner. Using the band saw, a coronal plane cutis made along the entire length of the spinal column at the anterioraspect of the pedicles leaving anterior tissues intact. Tissuesposterior to the disc space are discarded. Next, the anterior column ofthe spinal level is bisected mid-sagittally to produce right and lefthalves. The entire disc space is left intact. The inferior half of theL4 anterior column adjacent to the treated level is retained. Thesuperior half of the L5 anterior column adjacent to the treated level isretained. Right and left sagittal samples from the level are so labeled,fixed in formalin, and processed (sequentially dehydrated in alcohols,cleared in a xylene substitute, and embedded in graded catalyzed methylmethacrylate.

After polymerization is complete and the samples hardened, sectioningand staining is performed. The blocks containing the right and lefthalves of the treated aspect of the spinal level are sectioned in thesagittal plane on a low speed diamond saw (Buehler Isomet, Lake Bluff,Ill.). For all embedded tissue blocks, sagittal sectioning commence fromthe middle of the treated aspect of the spinal level to the lateralaspect of the treated area. Thus, section #1 from the “right block” issampled in the middle of the fusion mass whereas section #6 from the“right block” is sampled at the far lateral aspect of the treated area.Weights are used to produce sections on the order of 300 μm.Approximately 5-10 sections are made in the sagittal plane through eachhalf of the interbody space. If necessary, grinding is performed toobtain the desired thickness. The thickness of the sections is measuredwith a metric micrometer (Fowler, Japan). Differential staining using atrichrome stain is used to permit histological differentiation.

Stained undecalcified sections are scanned using image analysis software(Image Pro Plus Software v 5.0, Media Cybernetics, Silver Spring, Md.)running on a Windows XP workstation. A video camera (Model DFC 280,Leica Microsystems, Cambridge, UK) is used to acquire the digital imagesof the stained undecalcified sections.

Section Fusion Criteria:

Undecalcified sections are evaluated for fusion in the center of thedisc space or thrugrowth region of the device, in the anterior margin,and in the posterior margin. These anatomic locations for each sectionare considered to be fused only if continuous bony bridging is foundfrom superior to inferior.

Level Fusion Criteria:

Based on all sections evaluated, the following criteria are used todetermine if histologic fusion is present in the level. The spinal levelis considered fused if greater than 50% of the sections (correspondingmicroradiographs are analyzed concurrently but not “counted twice” forfusion) show continuous bony bridging. A partial fusion exists if lessthan 50% of the sections (and corresponding microradiographs) showcontinuous bony bridging. A non-fusion exists if none of the sectionsand corresponding microradiographs show continuous bony bridging.

Microradiography

Undecalcified sections from the treated lumbar spinal levels areradiographed using a microradiography unit (Faxitron radiography unit,Hewlett Packard, McMinnville, Oreg.) and spectroscopic film (B/RA 4153film, Kodak, Rochester, N.Y.). The thickness of the sections is measuredwith a metric micrometer (Fowler, Japan) to determine the exposure time.Sections are labeled with ultra-fine permanent markers, placed on theEktascan B/RA 4153 film, and exposed to the x-ray source at 20 kV and 3mA for approximately 45 seconds for each 100 μm of section thickness.The film is then developed, fixed, and analyzed for ossification usingstandard optical microscopy. Microradiographs are scanned using imageanalysis software (Image Pro Plus Software v 5.0, Media Cybernetics,Silver Spring, Md.) running on a Windows XP workstation. A video camera(Model DFC 280, Leica Microsystems, Cambridge, UK) is used to acquirethe digital images of the microradiographs.

Analysis of the sections and microradiographs is used to:

-   1) Evaluate the extent of fusion adjacent to and within the PEEK    spacers, bone graft and Example 3 Putty incorporation, and bone    remodeling within the fusion mass,-   2) Determine the host response to the biomaterials used, and-   3) Evaluate the interface of the PEEK spacer.

Example 5 Use of Osteogenic Putty in Posterolateral Fusion

An instrumented ovine posterolateral fusion model is used to evaluatethe ability of Autograft and the Osteogenic Putty of Example 3 to effectposterolateral fusion at 6 months post-operatively. The efficacy ofthese treatments to induce posterolateral fusion in the ovine lumbarfusion model is evaluated using blinded radiographic, biomechanical, andhistologic measures. Assessment of fusion is made with Faxitronhigh-resolution radiography, non-destructive biomechanical testing, andundecalcified histology with microradiography. All analyses areconducted in a blinded fashion. In addition, undecalcified histology isused to evaluate the osteocompatibility of the osteogenic putty. Inaddition to the treatment groups being evaluated, biomechanicalproperties of normal spines are evaluated using the same methodology.When all data acquisition is complete, the key is broken, andradiographic, biomechanic, and histologic data are analyzed by treatmentgroup.

Animal Model:

The sheep lumbar spine model is used because of the biomechanicalsimilarities between the sheep and human lumbar spine. Wilke et al.characterized the biomechanical parameters (range of motion, neutralzone, and level stiffness) of sheep spines and made comparisons withdata from human specimens previously published by White and Panjabi(see, Wilke et al., Spine: 22(20): 2365-2374, 1997; and White A A andPanjabi M M, editors, Clinical Biomechanics of the Spine, 2nd ed., J. B.Lippincott, Philadelphia, Pa., 1990). Wilke et al. found that the“ranges of motion of sheep spines for the different load directions arequalitatively similar in their craniocaudal trends to those of humanspecimens reported in the literature.” They further concluded that:“Based on the biomechanical similarities of the sheep and human spinesdemonstrated in this study, it appears that the sheep spine . . . canserve as an alternative for the evaluation of spinal implants.

Surgical Technique:

12 sheep are placed in the appropriate pastures of the large animalresearch barn. They are dewormed and eartagged for identification.Physical examination is performed and any animals with signs ofrespiratory disease have venous blood submitted for a complete bloodcount (CBC).

The sheep are anesthetized. Wool is removed from the dorsal lumbar areaand the sheep positioned in sternal recumbency on the operating table.The dorsal and dorsolateral lumbar area are prepared for aseptic surgerywith multiple scrubs of povidone-iodine alternated with isopropylalcohol. The area is draped and a dorsal approach to L3-L6 is madethrough the dorsal lumbar musculature.

Iliac Crest Autograft Harvesting:

Autograft is used as a control. The following protocol is followed. Thedorsal and dorsolateral lumbar and iliac crest areas are prepared foraseptic surgery with multiple scrubs of povidone-iodine alternated withisopropyl alcohol. The area is draped and a 3-cm incision made over theleft iliac crest. Following partial reflection of the gluteal muscles,an osteotome is used to create a small window in the craniodorsal faceof the iliac crest. Using a curette, about 2 cc of autogenous cancellousbone is removed, and is later packed into one of the implants (e.g. PEEKspacer) used for the lumbar fusion (this is the control case).Intralesional morphine sulfate is administered prior to closure of theiliac crest incision. The iliac crest site is closed routinely using 2/0polysorb for the subcutaneous tissues and stainless steel staples forthe skin.

Dorsolateral (“Posterolateral”) Interbody Fusion:

The dorsal lumbar area is prepared for aseptic surgery with multiplescrubs of povidone-iodine alternated with isopropyl alcohol. The area isdraped and local anesthesia (Bupivicaine) is infiltrated along the siteof the intended incision for the dorsal approach to L3 and L4 andspinous processes.

Approach to the Transverse Processes:

A 20 cm. skin incision is made and the paraspinal muscles are dissectedoff the spinous processes and laminae. Facet joints and transverseprocesses between L3 and L4 are exposed.

Instrumentation and Spine Fusion Technique:

The transverse processes of L3 and L4 are decorticated bilaterally. Thebone graft from the iliac crest or the osteogenic putty of Example 3 isplaced between the transverse processes (˜10 cc per side). The sheep nowundergoes transpedicular screw fixation using screws and rods. Thepedicle screws and rods are inserted at this point in the procedure.

Wound Closure:

Routine closure of external abdominal muscular fascia (0 Polysorb(absorbable suture), subcutaneous tissue (2/0 Polysorb and skin (2/0monofilament non-absorbable suture) is performed. Operative time foreach animal is about 50 minutes. Perioperative antibiotics (Cephazolinsodium) are administered. Postoperative radiographs are performed whilethe sheep are still under general anesthesia.

Aftercare:

Immediately after surgery, the sheep are transferred from the operatingtable to a modified wheelbarrow and while still under generalanesthesia, and taken to a radiology suite where dorsoventral andlateral radiographs of the fusion sites are obtained. Followingradiographic evaluation, while still in the modified wheelbarrow, theyare observed until the swallowing reflex returns. At that point they areextubated and taken to a trailer where they are propped in sternalrecumbency. At the end of the day, all animals that are operated uponthat day are moved to research pastures. The sheep are housed outdoors(with access to a three-sided shelter) for the convalescence and allowedto exercise at will. Postoperative analgesia is provided as described.The sheep are anesthetized and radiographed at three monthsposoperatively.

Euthanasia:

After 6 months postoperatively, the 12 sheep are euthanized in a humanemanner. Euthanasia is performed according to the guidelines set forth bythe AVMA Panel on Euthanasia (J. Am. Vet. Med. Assoc., 202:229-249,1993). Radiographs of the lumbar fusion sire are taken in these sheep toevaluate the degree of fusion at L3-L4.

Specimen Collection and Handling:

Following euthanasia, a complete gross necropsy is conducted on all 12animals. Conventional gross examination of all major organ systems andhistopathological evaluation of any pathological lesions is performed.Any animals that died or were prematurely euthanized during the courseof the study have a complete necropsy performed to determine the causeof disease or death. At necropsy the lumbar vertebrae that are fused areharvested.

Material Analysis:

All samples from the lumbar area from the sheep are subjected tomechanical testing of the fusion sites. They are tested for stiffness tosaggital and coronal plan bending moments (flexion, extension, right andleft lateral bending). As these mechanical tests are nondestructive, thefusion sites are also examined histologically.

Implant Materials:

The study groups are defined below.

Study Group (per study design) No. of Samples (N) 1) 10 cc/sideAutograft (Autograft) 6 2) Example 3 Putty 6 3) Normal Intact 17 Total28At the completion of the survival phase of the animal study, the spinesare immediately frozen for evaluation. The efficacy of the bone graftand bone graft substitutes to effect posterolateral fusion and bonyhealing is assessed by performing radiographic, biomechanical, andhistologic analyses as detailed below. The study is performed in ablinded fashion. After all analyses are completed, the key is broken andradiographic, biomechanical, and histologic data are analyzed bytreatment group.Methods of Analysis:1. Radiographic Assessment:

Radiographs are taken immediately after surgery, at regularpost-operative intervals, and at the time of sacrifice. A Faxitron(Hewlett Packard, McMinnville, Oreg.) high-resolution radiography unitand high-resolution film (EKTASCAN B/RA Film 4153, Kodak, Rochester,N.Y.) is used to produce a high-resolution PA radiograph of theharvested lumbar spines after biomechanical testing. Radiographs arescanned using image analysis software (Image Pro Plus Software v 5.0,Media Cybernetics, Silver Spring, Md.) running on a Windows XPworkstation. A video camera (Model DFC 280, Leica Microsystems,Cambridge, UK) is used to acquire the digital images of the radiographs.These radiographs are also used to gross the samples for histologicanalyses as outlined below.

Three blinded evaluators evaluate the resulting Faxitron radiographs forintertransverse process fusion. On the PA radiograph, on both the rightand left sides of the level, the intertransverse process space isevaluated for fusion based on the following scoring method: 4=continuousbony bridging, 3=increased bone density, 2=lucency with some bonybridging, and 1=non-fusion. Based on both the right and left sides ofthe PA radiographs, the blinded evaluators rate an overall fusion scorefor the spinal level using the following criteria:

3=Solid Fusion: Solid intertransverse process fusion on Right AND Leftwith no radiolucencies

2=Possible Fusion: Intertransverse process fusion on the Right OR Left,but not both. Lucencies in intertransverse process space on right orleft.

1=Non-Fusion: Isolated bone formation without continuous superior toinferior bony bridging on both right and left sides. Significant lucencywith no evidence of intertransverse process fusion on the right or left.

After the treatment code is broken, the radiographic fusion data arestatistically analyzed.

2. Ex-Vivo Biomechanical Testing of the Treated Lumbar Motion Segment:

Flexibility Testing:

Unconstrained biomechanical testing is performed in a non-destructivemanner on all spines after the frozen specimens are thawed. All metallicposterior instrumentation used to stabilize the posterolateral fusion isremoved prior to biomechanical testing so that the stiffness of thespine and fusion mass construct is tested. Flexibility of the motionsegments is determined in flexion, extension, right and left lateralbending, and right and left axial rotation. All tests are performedwithin 12 hours of specimen thawing. Specimens are only frozen once. Thepurpose of the biomechanical testing is to quantify the stiffness of thelumbar motion segments augmented with the previously described fusiontreatments. The treated (L4-L5) motion segments are dissected from theharvested lumbar spines and cleaned of extraneous soft tissue leavingthe ligamentous and osseous tissues intact. Specially designed loadingand base frames are secured on the L4 and L5 vertebra, respectively.

Moments of 0, 0.5, 2.5, 4.5, 6.5, and 8.5 Nm are achieved in eachloading direction. Static loads are used to apply the pure moments. Asix-degree of freedom load cell is placed in series with the testedspecimen to verify the applied moments. Three markers reflecting theinfrared light are attached to each vertebra. The locations of theinfrared reflective markers are recorded using three VICON cameras(Vicon Peak, Oxford, England) at each load. Three-dimensionalload-displacement data are then acquired with pure moments applied inflexion, extension, left and right lateral bending, and left and rightaxial rotation. The three-dimensional coordinate data are analyzed toobtain the rotation angles of the superior vertebra with respect to theinferior vertebra and rotational flexibility of each motion segment.

Biomechanics data from a normal (untreated) intact group of sheep lumbarspine motion segments that have been obtained previously are used asbaseline data for normal lumbar spine motion for L4-L5 in sheep.Differences in the stiffness (flexibility) between groups and thenormals are statistically compared. Non-parametric Kruskal-Wallis andMann-Whitney tests are used to analyze the biomechanics data.

3. Undecalcified Histology and Microradiography:

Processing and Stained Undecalcified Sections:

In each of the treatment groups, the bisected spinal intertransverseprocess spaces are analyzed using undecalcified techniques(microradiographs and multiple stain). Differential staining along withqualitative optical microscopy is performed to assess bony bridging andextent of fusion associated with autograft and the osteogenic putty ofExample 3.

After Faxitron radiography, all spinal levels containing an implant aregrossed in the following manner. The superior (L3-L4) and inferior(L5-L6) disc spaces are transected leaving the treated (L4-L5)functional spinal unit (FSU) intact. Using the band saw, a coronal planecut is made along the entire length of the spinal column at the anterioraspect of the pedicles leaving posterior tissues intact. Anteriortissues are discarded. Next, the posterior elements of the spinal levelare bisected mid-sagittally to produce right and left halves. An angledcut in the axial plane is made so that tissues cranial to the cranialtransverse processes are discarded on the right and left sides. Anangled cut in the axial plane is made so that tissues caudal to thecaudal transverse processes are trimmed and discarded on the right andleft sides. Tissues in the Right and Left intertransverse process spacesare further divided in the sagittal plane to produce a medial andlateral sample of the Left fusion mass as well as a medial and lateralsample of the Right fusion mass. Right and left medial and lateralsamples are so labeled, fixed in formalin, and processed (sequentiallydehydrated in alcohols, cleared in xylene or xylene substitute, andembedded in graded catalyzed methyl methacrylate).

After polymerization is complete and the samples hardened, sectioningand staining is performed. The blocks containing the transverseprocesses, autograft and Example 3 putty, and tissues in the transverseprocess space are sectioned in the sagittal plane on a low speed diamondsaw (Buehler Isomet, Lake Bluff, Ill.). For the medial and lateralembedded tissue blocks described above, sectioning commences from themiddle of the fusion mass for both the medial and lateral blocks. Thus,section #1 from the “right lateral block” is sampled in the middle ofthe fusion mass whereas section #6 from the “right lateral block” issampled at the far lateral anatomic aspect of the fusion mass (tips ofthe transverse processes). Similarly, section #1 from the “right medialblock” is sampled in the middle of the fusion mass whereas section #6from the “right medial block” is sampled at the far medial anatomicaspect of the fusion mass (lamina and facet joints). Weights are used toproduce sections on the order of 300 μm. Approximately 5-10 sections aremade in the sagittal plane through each half of the intertransverseprocess space. If necessary, grinding is performed to obtain the desiredthickness. The thickness of the sections is measured with a metricmicrometer (Fowler, Japan). Differential staining using a trichromestain is used to permit histological differentiation.

Stained undecalcified sections are scanned using image analysis software(Image Pro Plus Software v 5.0, Media Cybernetics, Silver Spring, Md.)running on a Windows XP workstation. A video camera (Model DFC 280,Leica Microsystems, Cambridge, UK) is used to acquire the digital imagesof the stained undecalcified sections. Undecalcified histology sectionsand microradiographs for this study are scanned so that dorsal is at thetop of the image. The ventral side of the section is usually flat andshows two oval transverse processes. Sections are scanned so thattransverse processes are at the bottom (ventral aspect) of the image. Amm scale is scanned at the bottom (ventral aspect) of the image.Microsoft Photo editor is used to crop the images.

Section Fusion Criteria:

Undecalcified sections are considered fused if continuous bony bridgingis found from superior to inferior in the section. If the presence ofnon-osseous tissues obviated continuous bony bridging, the section isfurther evaluated as follows. For non-fused sections, sections areclassified as A) non-fusion with incomplete bridge, but with de novobone found in >50% of the length of the section, or B) non-fusion withincomplete bridge, with de novo bone found in <50% of the length of thesection.

Right and Left Side Level Fusion Criteria:

Based on all sections evaluated, the following criteria are used todetermine if histologic fusion is present on the right or left side ofthe level. The right or left side of the spinal level is consideredfused if greater than 50% (>50%) of the sections and correspondingmicroradiographs show continuous bony bridging. A partial fusion existsif 50% or less ≤50%) of the sections and corresponding microradiographsfrom the right or left side of the spinal level show continuous bonybridging. A non-fusion exists if none of the sections and correspondingmicroradiographs from the right or left side of the spinal level showscontinuous bony bridging.

Microradiography:

Undecalcified sections from the treated lumbar spinal levels areradiographed using a microradiography unit (Faxitron radiography unit,Hewlett Packard, McMinnville, Oreg.) and spectroscopic film (B/RA 4153film, Kodak, Rochester, N.Y.). The thickness of the sections is measuredwith a metric micrometer (Fowler, Japan) to determine the exposure time.Sections are labeled with ultra-fine permanent markers, placed on theEktascan B/RA 4153 film, and exposed to the x-ray source at 20 kV and 3mA for approximately 45 seconds for each 100 μm of section thickness.The film is then developed, fixed, and analyzed for ossification usingstandard optical microscopy. Microradiographs are scanned using imageanalysis software (Image Pro Plus Software v 5.0, Media Cybernetics,Silver Spring, Md.) running on a Windows XP workstation. A video camera(Model DFC 280, Leica Microsystems, Cambridge, UK) is used to acquirethe digital images of the microradiographs.

Analysis of the sections and microradiographs is used to:

-   1) Evaluate histologic fusion,-   2) Determine the host response to the autograft and bone graft    substitutes, and-   3) Estimate the quality and quantity of bone in the fusion mass    within the intertransverse process space.

The uses of the terms “a” and “an” and “the” and similar references inthe context of describing the invention (especially in the context ofthe following claims) are to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiment has been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected. In addition, all references cited hereinare indicative of the level of skill in the art and are herebyincorporated by reference in their entirety.

What is claimed is:
 1. A method, comprising: providing an osteoinductiveimplant material comprising an osteoconductive scaffolding materialincorporating an osteoinductive protein; coating exterior surfaces ofthe osteoinductive implant material with a biodegradable barriersubstance to form a coated implant material, wherein said biodegradablebarrier substance is effective to delay release of the osteoinductiveprotein from the scaffolding material; and implanting said coatedimplant material in a patient at a site at which bone growth is needed,wherein said coating is conducted in a sterile operating field.
 2. Themethod of claim 1, wherein said osteoinductive protein comprises a bonemorphogenic protein, and wherein said scaffolding material exhibits acapacity to bind said bone morphogenic protein.
 3. The method of claim2, wherein said providing comprises incorporating said osteoinductiveprotein into said osteoconductive scaffolding material to prepare saidosteoinductive implant material, and wherein said incorporating isconducted in the sterile operating field.
 4. The method of claim 3,wherein sulfated glycosaminoglycan molecules are bound to saidscaffolding material, and wherein said bone morphogenic protein exhibitsa capacity to bind to said sulfated glycosaminoglycan molecules.
 5. Themethod of claim 1, further comprising binding sulfated glycosaminoglycanmolecules to said scaffolding material using end point binding, whereinsaid osteoinductive protein exhibits a capacity to bind to said sulfatedglycosaminoglycan molecules.
 6. The method of claim 5, wherein saidsulfated glycosaminoglycan molecules comprise at least one of heparinand dextran sulfate.
 7. The method of claim 1, further comprisingbinding sulfated glycosaminoglycan molecules to said scaffoldingmaterial using an aminosilane coupling agent, wherein saidosteoinductive protein exhibits a capacity to bind to said sulfatedglycosaminoglycan molecules.
 8. The method of claim 1, furthercomprising binding sulfated glycosaminoglycan molecules to saidscaffolding material by covalent crosslinking using glutaraldehyde as acoupling agent, wherein said osteoinductive protein exhibits a capacityto bind to said sulfated glycosaminoglycan molecules.
 9. The method ofclaim 1, further comprising treating surfaces of said scaffoldingmaterial by adding one of a negative surface charge and a positivesurface charge to increase binding of said osteoinductive protein tosaid scaffolding material.
 10. The method of claim 1, wherein saidproviding comprises incorporating sulfated glycosaminoglycan moleculeson a precursor material and processing said precursor material to formsaid scaffolding material, wherein said osteoinductive protein exhibitsa capacity to bind to said sulfated glycosaminoglycan molecules.
 11. Themethod of claim 1, further comprising ionically crosslinking alginateusing an ionic crosslinking agent comprising calcium chloride to formsaid biodegradable barrier substance.
 12. The method of claim 1, whereinsaid biodegradable barrier substance comprises a liquid organic binderthat includes ionic polysaccharides that are capable of formingthermally-irreversible ionically-crosslinked gels, and said methodfurther comprises implanting said coated implant material in a patientat a site at which bone growth is needed by co-administering said coatedimplant material with a liquid medium of an ionic crosslinker.
 13. Themethod of claim 12, wherein said co-administering comprises moving saidcoated implant material through a first barrel of a syringe and movingsaid liquid medium through a second barrel of said syringe such thatsaid coated implant material and said liquid medium are admixed as theyexit said syringe.
 14. The method of claim 1, wherein said biodegradablebarrier substance comprises a liquid organic binder that includes ionicpolysaccharides that are capable of forming thermally-irreversibleionically-crosslinked gels, and said method further comprises implantingsaid coated implant material in a patient at a site at which bone growthis needed by administering said coated implant material without an ioniccrosslinker.
 15. The method of claim 1, further comprising implantingsaid coated implant material in a patient at a site at which bone growthis needed by incorporating said coated implant material within aload-bearing spinal implant.
 16. The method of claim 15, wherein saidload-bearing spinal implant is a fusion cage.
 17. The method of claim15, wherein said load-bearing spinal implant has a compression strengthof at least 10,000 N.
 18. The method of claim 1, wherein said providingcomprises removing said osteoinductive implant material from a sterilepackage in a sterile operating field and said coating exterior surfacesof the osteoinductive implant material comprises applying saidbiodegradable barrier substance as a coating on said osteoinductiveimplant material while in the operating field.